Abstract
Four versions of tricistronic vectors expressing IgG1 light chain (LC), IgG1 heavy chain (HC), and dihydrofolate reductase (DHFR) in one transcript were designed to compare internal ribosome entry site (IRES) and furin-2A (F2A) for their influence on monoclonal antibody (mAb) expression level and quality in CHO DG44 cells. LC and HC genes are arranged as either the first or the second cistron. When using mAb quantification methods based on the detection antibodies against HC Fc region, F2A-mediated tricistronic vectors appeared to express mAb at higher levels than the IRES-mediated tricistronic vectors in both transient and stable transfections. Further analysis revealed that more than 40% of products detected in stably transfected pools generated using the two F2A-mediated tricistronic vectors were aggregates. LC and HC from the F2A stably transfected pools were not properly processed, giving rise to LC+F2A+HC or HC+F2A+LC fusion proteins, LC and HC polypeptides with F2A remnants, and incorrectly cleaved signal peptides. Both IRES-mediated tricistronic vectors express mAb with correct sizes and signal peptide cleavage. Arrangement of LC as the first cistron in the IRES-mediated tricistronic vectors exhibits increased mAb expression level, better growth, and minimized product aggregation, while arrangement of HC as first cistron results in low expression, slower growth, and high aggregation. The results obtained will be beneficial for designing vectors that enhance mAb expression level and quality in mammalian cells.
Highlights
Monoclonal antibodies are currently the fastest growing class of biotherapeutic molecules [1,2]
Design of internal ribosome entry site (IRES)- and F2A-mediated Tricistronic Vectors An anti-HER2 IgG1 biosimilar was first used as a model monoclonal antibody (mAb) to compare IRES and F2A for mAb expression level and quality
The two vectors in which light chain (LC) and heavy chain (HC) are linked by a wild type encephalomyocarditis virus (EMCV) IRES (IRESwt) are designated as LIRES-H and H-IRES-L, respectively
Summary
Monoclonal antibodies (mAbs) are currently the fastest growing class of biotherapeutic molecules [1,2]. MAbs are commonly produced by stable transfection of Chinese hamster ovary (CHO) cells with the HC, LC and selection marker on either one or two separate vectors [3,4,5,6]. Each gene is under the control of its own promoter and transcribed separately. One disadvantage of such designs is that vector fragmentation could result in non-expressing clones surviving drug selection [7,8]. The other disadvantage is the lack of control over the ratio of LC:HC expression. It has been demonstrated that expression of LC in excess was beneficial for mAb expression [10,11,12,13,14,15]. Having HC in excess can cause ER stress [17] and proteasome overloading [18], creating a burden to the cell machinery and can inhibit cell proliferation [15]
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